Methods and ProtocolsWe had a total of 8 experiments, each with it's own goals. Below are the protocols we followed.
miRNA and DNA Storage
miRNA strands and DNA probes were purchased from IDT as desalted solids. For the DNA, RNAase free water is added to make the concentration 13.33 µM (ready for ligation). The DNA was then aliquoted and stored at -20 ˚C. For the miRNA, TE buffer (Ambion, pH 8.0) was added to make final concentration 100 µM. The miRNA was then aliquoted and stored at -80 ˚C.
CircLigase ssDNA Ligase is a thermostable ATP-dependent ligase that catalyzes circularization of ssDNA templates with exposed 5´-phosphate and 3´-hydroxyl groups without the reliance on a complementary DNA sequence. 50 pmol of ssDNA probe initially designed with break in the loop region was ligated with 5U of ligase in 2 µL of 10X CircLigase Reaction Buffer and 50 mM MnCl2 in a total reaction volume of 20 µL. Ligation reaction took place at 60oC for 2 hours and the enzyme was heat inactivated at 80oC for 10 min.
With the design of a new DNA probe (custom-made and purchased from IDT), whose break site lies within its double stranded stem region, the more common and cost efficient ligation method using the T4 ligase was employed. The enzyme was purchased from ThermoFisher Scientific. It catalyzes the formation of phosphodiester bonds in the presence of ATP and hence the linking of DNA ends in the double-stranded region. 24 µL T4 DNA ligase buffer (composed of 250 mM Tris-HCl (pH 7.6), 50 mM MgCl2, 5mM ATP, 5mM DTT, 25% (w/v) polyethylene glycol-8000) was added to 90µL of 13.33 µM linear ssDNA probe. This was heated to 75 oC for 10 min (melting temperature of DNA probe was 70.7 oC), then cooled slowly to room temperature to establish desired oligomer conformation. 30 U of the T4 ligase was incubated with the linear DNA probe for 10-15 minutes to ligate the DNA for assay experiments. The enzyme was deactivated after ligation by incubating the mixtures at 65oC for 10 minutes.
Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)
Urea PAGE (10-15% acrylamide) was utilized to validate both methods of ligation, which allows for high resolution separation of denatured DNA fragments under 500 bps (see detailed procedures in protocol folder). The gels were ran for 50 minutes at 110 V. Then they were stained with SYBR Gold for 30 minutes and imaged using a transilluminator.
First, A master mix was made with: 10X phi29 reaction buffer (CutSmart buffer if subsequent steps included addition of restriction enzyme) from NEB, RNAase free water, 200 mM of dNTPs (Invitrogen), 100 nM of DNA probes and 4 U/sample of phi29. The mixture was vortexed for 5 seconds and added to 0.5 mL PCR tubes.
miRNA standards are prepared from a stock of 100 µM. First a 1e-6 M dilution is prepared in RNAase free water. Then this solution is serially diluted by a factor of 10 until sufficient dilution is achieved for the last sample in the experiment.
1.2 µL of each standard concentration of miRNA is added to its corresponding tube (final sample volume after addition of restriction enzymes and SYBR Gold is 120 µL). The tubes are incubated in a 30oC room for 2 hours. The tubes are then incubated at 65 oC in a heating block for 10 minutes to denature the enzyme and then for 10 minutes at room temperature to bring temperature down. 20 U of HAEIII restriction enzyme (NEB) is then added each tube and the tubes are incubated at 37 oC for 30 minutes. After that, HAEIII is denatured by incubateion at 80 oC for 20 minutes. 1.2 µL of 100X SYBR Gold (Thermo Scientific) is added to each tube. The tubes are mixed 100 µL from each tube is added to a fluorescence assay plate. The signal is measured with excitation at 495 nm and emission at 538 nm.
For the selectivity test, another miRNA strand was designed where it had 3 nucleotides different from miR-193b:
Correct Sequence: 5’-AACUGGCCCUCAAAGUCCCGCU-3’
Variant Sequence: 5’-AACUGGCCCUCAAAGTCTCTCU-3’
All other conditions were kept the same for both miRNA sequences.
For the final experiment, higher concentrations of phi29 are used in a modified master mix that is expected increase our sensitivity . The master mix is made with 0.1 µM DNA, 10X phi29 reaction buffer, RNAase free water, 500 mM of dNTPs, 100 µM of BSA and 5 U of phi29. The mixture is vortexed for 5 seconds. miRNA standards are prepared from a stock of 100 µM, following the same method used in previous assay experiments. miRNA concentrations of 10-8 to 10-14and of 0 are prepared.
48µL of the master mix is added to 24 1.5mL labeled PCR tubes. 2 µL of each standard concentration of miRNA is added to three tubes. The tubes are incubated in a 30oC room for 2 hours to allow the RCA to occur. The tubes are then incubated at 65 oC in a heating block for 10 minutes to denature the enzymes. The tubes are cooled to room temperature.
10 U of HAEIII restriction enzyme is added to each tube. Tubes are incubated at 37 oC for 30 minutes then 80 oC for 20 minutes. Tubes are cooled to room temperature. 1 µL of 100X SYBR Gold is added to each tube and the tubes are covered to incubate at room temperature for 5 minutes. 68 µL of RNase free water is added to all tubes with miRNA and 70 µL is added to three tubes with no miRNA.The tubes are mixed 100 µL from each tube is added to a fluorescence assay plate. The signal is measured with excitation at 495 nm and emission at 538 nm.